Abstract Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by the production of antinuclear antibodies (ANAs) in association with severe multisystem inflammatory disease manifestations. ANAs target a wide array of nuclear macromolecules and can mediate disease by the formation of immune complexes that deposit in the tissue to promote inflammation and damage; these complexes can also stimulate the production of cytokines including type 1 interferon to drive generalized immune system disturbances. Among ANAs forming immune complexes, antibodies to DNA (anti-DNA) are the serological hallmark of SLE and markers of diagnosis and prognosis. These antibodies bind to both single and double stranded DNA and form immune complexes that deposit in the tissue, especially the kidney, to promote inflammation; in addition, these immune complexes can stimulate the production of cytokines by plasmacytoid dendritic cells, triggering internal sensors of DNA. While the properties of anti-DNA antibodies have been extensively studied, much less is known about the origin of antigenic DNA, its access to the immune system and its unique molecular properties. In general, the source of this DNA has been considered to be nuclear in origin and the product of dead and dying cells. Our studies have provided a new perspective on this issue by demonstrating two important features of extracellular DNA: 1) its existence in the form of microparticles and 2) the presence in these particles of mitochondrial (mt) as well as nuclear DNA (nDNA). This presence of mtDNA is important since mtDNA is intrinsically immunostimulatory because of structural features that differ from those of nDNA. These involve CpG motifs in mtDNA, a consequence of the origin of mitochondria from bacteria, and oxidation of guanosine residues. Furthermore, we have shown that mitochondria released from cells have properties similar to those of microparticles, suggesting that mitochondria may serve as self-antigens in lupus, contributing to immune activities attributed to microparticles and providing a nidus for immune complex formation. Building on preliminary work, the proposed studies will focus on three main hypotheses: 1) DNA autoantigen can be released from cells undergoing various death forms and exist in both a free and particle form; 2) the metabolism of DNA during cell death influences the amount in the blood, its size and its representation in particle or soluble form; and 3) anti-DNA antibodies can bind to various antigenic forms of DNA, both free and particulate, with assay of antibodies to these forms providing more informative biomarkers. We will pursue three specific aims. Specific Aim 1: To determine the extracellular release of cellular DNA during different forms of in vitro cell death. We will induce different forms of cell death in cell lines in vitro and determine the representation of mtDNA and nDNA in soluble and particulate forms; Specific Aim 2. To use in vivo models to elucidate the release of DNA into the blood. We will use in vivo systems in the mouse to explore the release of mtDNA and nDNA following cell death; we will also sequence DNA in the blood by high throughput techniques; and Specific Aim 3: Using blood from patients with lupus and murine models, we will determine the presence and specificity of antibodies to various sources of extracellular DNA in SLE. To develop new serological approaches, we will determine the presence and specificity of antibodies to different antigenic forms of DNA, both free and particulate and, by assessing results from a large panel of well characterized patients, develop new biomarkers. Successful completion of these experiments will provide new insights into the mechanisms of pathogenicity as well as provide new biomarkers for clinical and research purposes.